Environmental Impact Evaluation for Heterogeneously Catalysed Starch Oxidation.

Oxidised starch is currently produced from native starch using sodium hypochlorite as an oxidising agent. The use of hypochlorite has undesired side reactions and produces stoichiometric amounts of waste (salt), thus alternative oxidation methods are desired. In this study, the potential of two catalysed starch oxidation methods to reduce the environmental impact (EI) of oxidised starch production are assessed. We compared the EI of oxidation with molecular oxygen (heterogeneously catalysed) and hydrogen peroxide (homogeneously catalysed) to hypochlorite oxidation through life cycle assessment (LCA). The results confirm that hypochlorite oxidation is the main environmental hotspot in the current process of oxidised starch production, and that both hydroperoxide oxidation and molecular oxygen oxidation can significantly lower the EI of the process. The impact reduction is most significant in the categories of freshwater eutrophication (∼67 %), ozone depletion (∼66 %), climate change (35-60 %) and resource use (40 %-78 %) for peroxide and molecular oxygen oxidation, respectively.

Maize feedstocks with improved digestibility reduce the costs and environmental impacts of biomass pretreatment and saccharification.

BACKGROUND: Despite the recognition that feedstock composition influences biomass conversion efficiency, limited information exists as to how bioenergy crops with reduced recalcitrance can improve the economics and sustainability of cellulosic fuel conversion platforms. We have compared the bioenergy potential-estimated as total glucose productivity per hectare (TGP)-of maize cultivars contrasting for cell wall digestibility across processing conditions of increasing thermochemical severity. In addition, exploratory environmental impact and economic modeling were used to assess whether the development of bioenergy feedstocks with improved cell wall digestibility can enhance the environmental performance and reduce the costs of biomass pretreatment and enzymatic conversion. RESULTS: Systematic genetic gains in cell wall degradability can lead to significant advances in the productivity (TGP) of cellulosic fuel biorefineries under low severity processing; only if gains in digestibility are not accompanied by substantial yield penalties. For a hypothetical maize genotype combining the best characteristics available in the evaluated cultivar panel, TGP under mild processing conditions (~3.7 t ha(-1)) matched the highest realizable yields possible at the highest processing severity. Under this scenario, both, the environmental impacts and processing costs for the pretreatment and enzymatic saccharification of maize stover were reduced by 15 %, given lower chemical and heat consumption. CONCLUSIONS: Genetic improvements in cell wall composition leading to superior cell wall digestibility can be advantageous for cellulosic fuel production, especially if "less severe" processing regimes are favored for further development. Exploratory results indicate potential cost and environmental impact reductions for the pretreatment and enzymatic saccharification of maize feedstocks exhibiting higher cell wall degradability. Conceptually, these results demonstrate that the advance of bioenergy cultivars with improved biomass degradability can enhance the performance of currently available biomass-to-ethanol conversion systems.

Quantitative modeling and analytic assessment of the transcription dynamics of the XlnR regulon in Aspergillus niger.

BACKGROUND: Transcription of genes coding for xylanolytic and cellulolytic enzymes in Aspergillus niger is controlled by the transactivator XlnR. In this work we analyse and model the transcription dynamics in the XlnR regulon from time-course data of the messenger RNA levels for some XlnR target genes, obtained by reverse transcription quantitative PCR (RT-qPCR). Induction of transcription was achieved using low (1 mM) and high (50 mM) concentrations of D-xylose (Xyl). We investigated the wild type strain (Wt) and a mutant strain with partial loss-of-function of the carbon catabolite repressor CreA (Mt). RESULTS: An improved kinetic differential equation model based on two antagonistic Hill functions was proposed, and fitted to the time-course RT-qPCR data from the Wt and the Mt by numerical optimization of the parameters. We show that perturbing the XlnR regulon with Xyl in low and high concentrations results in different expression levels and transcription dynamics of the target genes. At least four distinct transcription profiles were observed, particularly for the usage of 50 mM Xyl. Higher transcript levels were observed for some genes after induction with 1 mM rather than 50 mM Xyl, especially in the Mt. Grouping the expression profiles of the investigated genes has improved our understanding of induction by Xyl and the according regulatory role of CreA. CONCLUSIONS: The model explains for the higher expression levels at 1 mM versus 50 mM in both Wt and Mt. It does not yet fully encapsulate the effect of partial loss-of-function of CreA in the Mt. The model describes the dynamics in most of the data and elucidates the time-dynamics of the two major regulatory mechanisms: i) the activation by XlnR, and ii) the carbon catabolite repression by CreA.

Evaluation of design strategies for time course experiments in genetic networks: case study of the XlnR regulon in Aspergillus niger.

One of the challenges in genetic network reconstruction is finding experimental designs that maximize the information content in a data set. In this paper, the information value of mRNA transcription time course experiments was used to compare experimental designs. The study concerns the dynamic response of genes in the XlnR regulon of Aspergillus niger, with the goal to find the best moment in time to administer an extra pulse of inducing D-xylose. Low and high D-xylose pulses were used to perturb the XlnR regulon. Evaluation of the experimental methods was based on simulation of the regulon. Models that govern the regulation of the target genes in this regulon were used for the simulations. Parameter sensitivity analysis, the Fisher Information Matrix (FIM) and the modified E-criterion were used to assess the design performances. The results show that the best time to give a second D-xylose pulse is when the D-xylose concentration from the first pulse has not yet completely faded away. Due to the presence of a repression effect the strength of the second pulse must be optimized, rather than maximized. The results suggest that the modified E-criterion is a better metric than the sum of integrals of absolute sensitivity for comparing alternative designs.

d-Xylose concentration-dependent hydrolase expression profiles and the function of CreA and XlnR in Aspergillus niger.

Aspergillus niger is an important organism for the production of industrial enzymes such as hemicellulases and pectinases. The xylan-backbone monomer, d-xylose, is an inducing substance for the coordinate expression of a large number of polysaccharide-degrading enzymes. In this study, the responses of 22 genes to low (1 mM) and high (50 mM) d-xylose concentrations were investigated. These 22 genes encode enzymes that function as xylan backbone-degrading enzymes, accessory enzymes, cellulose-degrading enzymes, or enzymes involved in the pentose catabolic pathway in A. niger. Notably, genes encoding enzymes that have a similar function (e.g., xylan backbone degradation) respond in a similar manner to different concentrations of d-xylose. Although low d-xylose concentrations provoke the greatest change in transcript levels, in particular, for hemicellulase-encoding genes, transcript formation in the presence of high concentrations of d-xylose was also observed. Interestingly, a high d-xylose concentration is favorable for certain groups of genes. Furthermore, the repressing influence of CreA on the transcription and transcript levels of a subset of these genes was observed regardless of whether a low or high concentration of d-xylose was used. Interestingly, the decrease in transcript levels of certain genes on high d-xylose concentrations is not reflected by the transcript level of their activator, XlnR. Regardless of the d-xylose concentration applied and whether CreA was functional, xlnR was constitutively expressed at a low level.

Modeling and analysis of the dynamic behavior of the XlnR regulon in Aspergillus niger.

BACKGROUND: In this paper the dynamics of the transcription-translation system for XlnR regulon in Aspergillus niger is modeled. The model is based on Hill regulation functions and uses ordinary differential equations. The network response to a trigger of D-xylose is considered and stability analysis is performed. The activating, repressive feedback, and the combined effect of the two feedbacks on the network behavior are analyzed. RESULTS: Simulation and systems analysis showed significant influence of activating and repressing feedback on metabolite expression profiles. The dynamics of the D-xylose input function has an important effect on the profiles of the individual metabolite concentrations. Variation of the time delay in the feedback loop has no significant effect on the pattern of the response. The stability and existence of oscillatory behavior depends on which proteins are involved in the feedback loop. CONCLUSIONS: The dynamics in the regulation properties of the network are dictated mainly by the transcription and translation degradation rate parameters, and by the D-xylose consumption profile. This holds true with and without feedback in the network. Feedback was found to significantly influence the expression dynamics of genes and proteins. Feedback increases the metabolite abundance, changes the steady state values, alters the time trajectories and affects the response oscillatory behavior and stability conditions. The modeling approach provides insight into network behavioral dynamics particularly for small-sized networks. The analysis of the network dynamics has provided useful information for experimental design for future in vitro experimental work.

Scaling-up vaccine production: implementation aspects of a biomass growth observer and controller.

This study considers two aspects of the implementation of a biomass growth observer and specific growth rate controller in scale-up from small- to pilot-scale bioreactors towards a feasible bulk production process for whole-cell vaccine against whooping cough. The first is the calculation of the oxygen uptake rate, the starting point for online monitoring and control of biomass growth, taking into account the dynamics in the gas-phase. Mixing effects and delays are caused by amongst others the headspace and tubing to the analyzer. These gas phase dynamics are modelled using knowledge of the system in order to reconstruct oxygen consumption. The second aspect is to evaluate performance of the monitoring and control system with the required modifications of the oxygen consumption calculation on pilot-scale. In pilot-scale fed-batch cultivation good monitoring and control performance is obtained enabling a doubled concentration of bulk vaccine compared to standard batch production.

Online automatic tuning and control for fed-batch cultivation.

Performance of controllers applied in biotechnological production is often below expectation. Online automatic tuning has the capability to improve control performance by adjusting control parameters. This work presents automatic tuning approaches for model reference specific growth rate control during fed-batch cultivation. The approaches are direct methods that use the error between observed specific growth rate and its set point; systematic perturbations of the cultivation are not necessary. Two automatic tuning methods proved to be efficient, in which the adaptation rate is based on a combination of the error, squared error and integral error. These methods are relatively simple and robust against disturbances, parameter uncertainties, and initialization errors. Application of the specific growth rate controller yields a stable system. The controller and automatic tuning methods are qualified by simulations and laboratory experiments with Bordetella pertussis.